Quenching In Fluorimetry.

In this slide contains types of Quenching and its applications.
Presented by: K. Sandhya Rani (Department of pharmaceutical analysis).
RIPER,anantpur.

1. 1 Presented by Ms. K. Sandhya Rani (Reg.No:20L81S0702 ) Under the guidance of Dr. P.Ramalingam M.Pharm , P h.D., Director – R&D Division & Professor of Pharmaceutical and Medicinal Chemistry, President – IPA Local Branch – Anantapuramu . Quenching in Fluorimetry A Seminar as a part of curricular requirement for 1 st year M . Pharm 1 st semester

2. 2 Introduction Quenching Types of Quenching Applications Conclusion References Contents:

3. 3 Fluorescence: When a molecule absorbs light in the visible or ultraviolet range of the spectrum, it is excited from the electronic ground state to an excited state. It return to the ground state by releasing the absorbed energy in the form of heat. The emitted light is called Fluorescence. Introduction:

4. 4

5. 5 Quenching refers to any process that decreases the fluorescence intensity of a given substance. Eg :- pH, concentration, temperature, viscosity, presence of oxygen, heavy metals or specific chemical substances etc. Quenching takes place due to presence of an anion or ions with loosely bound electrons. During Quenching there is no permanent reaction between the fluorescent substance and the quenchers. Quenching:

6. 6 Quenchers are substances capable of absorbing energy from a fluorophore (such as a fluorescent dye) and re-emitting much of that energy as either heat (in the case of dark quenchers) or visible light (in the case of fluorescent quenchers). Eg : Molecular oxygen, Thiocyanate , Halogens, Disulfide, Nitric oxide. Quenchers:

7. 7 Fluorophores are fluoroscent chemical compounds that can re-emit light upon excitation. It absorbs light energy of specific wavelength and re-emits at longer wavelength. Fluorophores :

8. 8 Concentration Quenching Chemical Quenching Static Quenching Collisional Quenching Types of Quenching:

9. 9 Concentration Quenching may be caused by excessive absorption of either primary or fluorescent radiation by the solution. This is also called as Inner Filter effect. It is a kind of Self Quenching. It can be calculated by following equation: F=QI act 1.Concentration Quenching:

10. 10 In the above equation, Q = Fluorescence efficiency (Constant for a particular substance) I = Incident light (Constant for a instrument) a = Molecular extinction coefficient, which is constant for a substance t = Path length c = Concentration of the substance

11. 11 Deviations at higher concentrations can be attributed to self-quenching. Concentration of the Solution

12. 12 Chemical quenching is due to various factors like changes in pH, Presence of oxygen, halides and electron withdrawing groups, heavy metals etc., PH: Aniline at pH 5 to 13 gives blue fluorescence when excited at 290nm. But at pH <5 and >13 it does not exhibit fluorescence. Oxygen: Presence of oxygen leads to quenching because of its paramagnetic property. 2. Chemical Quenching:

13. 13 Halides and electron withdrawing groups: Halides like chlorine, bromine, iodide and electron withdrawing groups like nitro and carboxylic group leads to quenching. 3 . Static Quenching: It occurs at the ground state of Fluorescing molecule. Also called Contact quenching

14. 14 It involves the formation of chemical complex between the fluorophore and quencher. Ex: Caffeine and related xanthines and purines reduce intensity of riboflavin by static mechanism.

15. 15 It occurs by the interaction of a quencher molecule with an excited molecule of the fluorescing substance. Also called Dynamic quenching. Ex: Halides such as chloride or, iodides are well know collisional quenchers. 4. Collisional Quenching:

16. 16 Determine Membrane permeability to the quenchers. Determination of diffusion constant. Investigation of conformational states of proteins. Used as a indicator of DNA hybridization. Applications:

17. 17 Quenching is an undesirable effect an the possibility of encountering this type of interference should be evaluated in developing a fluorometric assay. However, this phenomenon can be used as an analytical means for determining the concentration of the compounds known to quench fluorescence . Conclusion:

18. 18 Douglas A Skoog , Donald M west, F James holler, Stanley R crouch, Molecular fluorescence spectroscopy, Text book of Fundamentals of analytical chemistry;8: 826-838. J. R. Lakowicz , Principles of Fluorescence Spectroscopy, 3rd ed., Springer, New York;2006: 954. Kautsky H, Quenching of luminescence by oxygen. Trans Faraday Soc;35: 216–219. Kommu Naresh , Applications of Fluorescence spectroscopy, journal of chemical and Pharmaceutical sciences;2004: 18-21. References:

19. 19 Thank You